Airway hyper-responsiveness (AHR) is a characteristic of asthma and COPD and results from the excessive contraction of airway smooth muscle cells (ASMCs). The exact cause of this excessive contraction is unknown but may involve changes in ASMC Ca2+ signaling or ASMC sensitivity to Ca2+. We have investigated the underlying mechanisms of Ca2+ signaling and Ca2+ sensitivity in order to better define mechanisms of AHR. We have investigated these processes in ASMCs surrounding small airways in lung slices made from the lungs of mouse, rats and humans. Isolated lungs are inflated with warm liquid agarose. When solidified by cooling, the agarose stiffens the lung tissue to allow slices, 250 um thick, to be cut. These slices are examined by phase-contrast microscopy to follow airway contraction as well as by confocal microscopy to observe ASMC Ca2+ signaling. We have found that methylcholine (from 100 - 1000 nM) induces Ca2+ oscillations in ASMC with frequencies ranging from 8 min-1 in humans to 20 min-1 in mice (at 1000 nM). In all cases, the extent of airway contraction increased in proportion to the increase the frequency of the Ca2+ oscillations. In human lung tissue, histamine induced similar changes (500 nm histamine induce a decrease in area to 63.98% +/- 7.88 and Ca2+ oscillations with a frequency of 5.73+/- 0.66 per min). These Ca2+ oscillations appeared to be primarily mediated by inositol-trisphosphate receptors (IP3Rs) because the addition of ryanodine, an antagonist of the ryanodine receptor, had no effect on agonist-induced Ca2+ oscillation frequency. Interestingly, the relationship of airway contraction with respect to the Ca2+ oscillation frequency was different between the 3 species. We developed an assay to evaluate Ca2+ sensitivity of ASMCs using caffeine and ryanodine to empty Ca2+ internal store and “clamp” internal Ca2+. With this preparation, we found that mouse ASMCs have a low Ca2+ sensitivity as compared to rat and human airways. In conclusion, ASMC contraction is a function of the frequency of Ca2+ oscillations, driven by the IP3R, and the level of Ca2+ sensitivity.